2.1 Field of the Invention
The invention relates to methods for identifying agents that modulate the catalytic activity and/or stability of mammalian DNA primase, compositions of such agents, improved DNA primase assay methods suitable for high-throughput screening to identify such agents, and the use of such methods to identify pharmaceutical agents and laboratory reagents which modify DNA primase activity.
2.2 The Related Art
DNA polymerase .alpha. and DNA primase are components of the DNA replication machinery, and play a role in the initiation of synthesis. In the double-stranded DNA of a chromosome, initiation occurs on both the leading and lagging strands, occurring more frequently on the lagging strand. The initiation of new DNA strands at origins of replication in animal cells involves the de novo synthesis of RNA primers by a primase activity and subsequent elongation of the RNA primers by one or more DNA polymerase activities, including that of DNA polymerase.
DNA primase initiates DNA replication by the synthesis of ribonucleotide primers which serve as a substrate for elongation by DNA polymerase a activity. The primase activity synthesizes short oligoribonucleotides during initiation of DNA replication and elongation of the lagging strand. DNA polymerase .alpha. elongates the RNA primer to complete the synthesis of the Okazaki fragment (Stillman B (1989) Ann. Rev. Cell. Biol. 5: 197). Okazaki fragments are then extended by either DNA polymerase .delta. or .epsilon., allowing DNA polymerase .alpha. and DNA primase to recycle and initiate another Okazaki fragment on the lagging strand (Waga S and Stillman B (1994) Nature 369: 207). This essential role of DNA primase makes it a key component in the regulation of initiation of DNA replication. A general overview of eukaryotic DNA replication is provided in Wang TSF (1991) Ann. Rev. Biochem. 60:513.
A unique property of DNA primase (as well as RNA polymerase) is the ability to synthesize oligonucleotides de novo on a template by the formation of an initial dinucleotide. DNA primase initiates synthesis with a triphosphate purine moiety at the 5' end (Gronostajski et al. (1984) J. Biol. Chem. 259: 9479). After synthesis of 7-10 ribonucleotides, the primer template is translocated intramolecularly to the active site of the DNA polymerase .alpha. subunit.
Mammalian DNA primase and DNA polymerase .alpha. copurify as a complex containing four subunits with approximate molecular masses of 180, 68, 58, and 49 kD. The 58 kD (p58) and 49 kD (p49) subunits can be separated from the 180 kD (p180) and 68 kD (p68) subunits and retain DNA primase activity when present as a p49-p58 complex. Thus, the p49 and p58 polypeptides are conventionally referred to in the art as the small subunit and large subunit of DNA primase, respectively.
The large and small subunits of DNA primase have been cloned as cDNA from mouse (Prussak et al. (1989) J. Biol. Chem. 264: 4957; Stadlbauer et al. (1994) Eur. J. Biochem. 222:781; Miyazawa et al. (1993) J. Biol. Chem. 268: 8111) and human (Stadlbauer et al. (1994) op.cit; Miyazawa et al. (1993) op.cit) and the sequences of the genes, gene products, coding nucleic acids and corresponding amino acid sequences of the proteins are reported in the EMBL/GenBank computer-accessible public databases, among others. Enzymatically active DNA primase has been produced from recombinant expression constructs in baculovirus-infected insect cells, yeast cells, and in E. coli cells (Bruckner et al. (1995) Mol. Cell. Biol. 15: 1716; Copeland WC and Wang TSF (1993) J. Biol. Chem. 268: 26179; Santocanale et al. (1992) Gene 113: 199). Alanine scanning mutagenesis has been performed to localize the catalytic site of the small subunit of mouse DNA primase (Copeland WC and Tan X (1995) J. Biol. Chem. 270: 3905).
The pharmaceutical sciences have identified agents which inhibit eukaryotic DNA replication at one or more steps. DNA replication inhibitory agents are used as human and veterinary drugs, such as antineoplastic agents (e.g., arabinosylcytosine, thioguanine, 5-fluorouracil, hydroxyurea, mitomycin, daunorubicin, doxyrubicin, actinomycin D, cyclophosphamide, etc.), antiviral agents (e.g., AZT, 3TC, ddI, acyclovir, gancyclovir, foscarnet, etc.), antifungal agents, and the like. Identification and development of new modulators of DNA replication and/or DNA repair synthesis provide new, improved, and/or alternative pharmaceuticals to treat diseases such as cancer, viral diseases (e.g., hepatitis B, AIDS), and other pathological conditions.
Arabinofuranosyl nucleotides have been reported to inhibit DNA synthesis by acting as chain terminators of elongation by DNA polymerase .alpha.. These same nucleotide "chain terminator" compounds have been reported to be incorporated by DNA primase and not result in chain termination (Thompson HC and Kuchta RD (1995) Biochemistry 34: 11198; Kuchta et al. (1992) Biochemistry 31: 4720). Thus, compounds which are known chain terminators of DNA polymerases are not necessarily chain terminators of DNA primase.
It would be advantageous to have a method for identifying agents which produce a decrease or, alternatively, an increase in mammalian DNA primase activity. Such DNA primase modulators could serve as candidate pharmaceutical agents to treat a variety of diseases, as well as laboratory reagents, for example as controls in a screen for primase activity, or for agents that modulate primase activity. Because of the large number of potential agents which can be screened for the activity of modulating DNA primase activity, it would be desirable if such a method were suitable for high-throughput screening of compound libraries. It would also be desirable to obtain compositions of specific DNA primase modulators, particularly agents which selectively modify DNA primase activity as compared to any effects on other enzymes involved in DNA metabolism (e.g., DNA polymerase (.alpha., .beta., .gamma., .delta., .epsilon.), helicase or telomerase) and/or RNA metabolism (e.g., RNA polymerase (pol I, pol II, pol III) and related proteins). As many current DNA synthesis/replication inhibitors are nucleotides, nucleosides, and analogs thereof, and much scrutiny is directed at developing this class of agents, it would be desirable to have a method suitable for identifying DNA primase modifiers which are compounds other than, or in addition to nucleotides or nucleosides.
The present invention fulfills these and other needs, and provides methods which will find wide applicability in the art.